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Purification of immunoglobulins using affinity chromatography and peptide ligands

a technology of affinity chromatography and immunoglobulin, which is applied in the field of purification of immunoglobulins using affinity chromatography and peptide ligands, can solve the problems of contaminating products, low stability, and high cos

Active Publication Date: 2006-07-13
NORTH CAROLINA STATE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018] A still further aspect of the invention is a method of identifying small peptide ligands with high affinity for immunoglobulins of interest, the method comprising the steps of: (a) screening a library of peptides, wherein the peptides are attached to a solid support, and wherein the peptides consist essentially of from 5 to 7 amino acid residues; (b) contacting a labeled immunoglobulin or labeled fragment thereof to the peptides; and then (c) determining the identity of the peptides bound to the the labeled immunoglobulin or fragment thereof.

Problems solved by technology

However, several drawbacks, including their high cost, low stability, and the possibility of contaminating products, make it important to find a new ligand for IgG purification.

Method used

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  • Purification of immunoglobulins using affinity chromatography and peptide ligands
  • Purification of immunoglobulins using affinity chromatography and peptide ligands
  • Purification of immunoglobulins using affinity chromatography and peptide ligands

Examples

Experimental program
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example 1

Peptide Synthesis

[0084] The peptide libraries used in this study were synthesized by Peptides International (Louisville, Ky.) on Toyopearl AF-amino-650 EC resin (Tosoh Bioscience, Montgomeryville, Pa.). The base resin (FIG. 2) from Tosoh Bioscience is a hydroxylated polymethacrylate amino resin with a pore size of 1000 Å and a particle size of 100-300 μm. The large particle size is particularly advantageous for use in screening since it makes it easier to identify and sequence the particles. Fmoc-L-alanine, a protecting group, was coupled to the amino functionality on the resin. Subsequently, the amino functionalities protected by Fmoc groups were released and used to link to other amino acids. Eighteen of the twenty amino acids (excepting cysteine and methionine) were used in this library synthesis. The amino acid lengths used were six (hexameric library), three (trimeric library) or one (monomeric library) and the peptide density was 400 μmoles / gm. The structure of this library i...

example 2

Positive Controls Used in Screenings

[0086] Two positive controls were used in this study to compare HIgG binding properties. They are protein A Sepharose CL-4B (protein A for short) and MAbsorbent A2P (A2P for short). Protein A Sepharose CL-4B (Amersham Biosciences, Piscataway, N.J.) is protein A immobilized by the CNBr method to 4% cross-linked agarose beads (Sepharose CL-4B) with a mean size of 90 μm. Protein A binds only to the Fc fragment of HIgG 1,2 and 4. MAbsorbent A2P (Prometic Biosciences, Burtonsville, Md.) is made by immobilizing a synthetic triazine based bifunctional ligand onto a 6% cross-linked agarose matrix (PuraBead 6XL) whose mean particle size is around 100 μm. MAbsorbent A2P binds to both Fc and Fab fragment as well as all sub-classes of IgG.

example 3

Radiolabeling of Whole HIgG and Fc Fragment of HIgG

[0087] Radiolabeling process. Fc fragment and HIgG (Bethyl Laboratories, Inc. Montgomery, Tex.) were labeled by reductive methylation (Equation 1, below) utilizing sodium cyanoborohydride (NaCNBH3) and 14C-formaldehyde (H14CHO, PerkinElmer) (Jentoft and Dearborn, 1983). The 14C formaldehyde was added at a 3-fold molar excess over 5% of the total methylation sites (39 total in Fc and more than 85 in intact HIgG). Sodium cyanoborohydride was added at 10× the amount of formaldehyde. The reaction was performed at 4° C. overnight. Following the reaction, the labeled protein was separated from 14C-formaldehyde using an EconoPac 10DG Desalting Column (Biorad, Hercules, Calif.) equilibrated with 0.1 M sodium phosphate, pH 7.0. The radioactivity of each fraction was determined using a Pacard 1500 Tri-Carb Liquid Scintillation Analyzer (Meridan, Conn.) and CytoScint ES scintillation liquid from ICN (Mesa, Calif.). The protein concentration i...

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Abstract

An immunoglobulin binding peptide having the general formula, from amino terminus to carboxy terminus, of Z-R1—R2—R3—R4—R5—R6—X, is described, wherein: R1 is H or Y; R2 is a hydrophobic, preferentially aromatic, amino acid (for example W, F, Y, V); R3 is a positively charged or aromatic amino acid (for example R, H, F, W); R4 is a hydrophobic or positively charged amino acid (for example G, Y, R, K, L); R5 is a positively charged or aromatic amino acid (for example W, F, R, H, Y); R6 a random amino acid but preferably hydrophobic or negatively charged (for example V, W, L, D, H); X is present or absent and when present is a linking group; and Z is present or absent and when present is a capping group bonded to the N terminus of R1; and wherein the amino acids of said peptide are in D form, L form, or a combination thereof. Methods of using such peptides for the purification of Immunoglobulins are also described

Description

FIELD OF THE INVENTION [0001] This invention concerns the making and use of peptide ligands for the purification of immunoglobulins from liquid containing immunoglobulins such as blood or blood plasma, plasma fractions, ascites fluid, aqueous cell culture, milk, and colostrums. BACKGROUND OF THE INVENTION [0002] The human immunoglobulins (Igs), a class of plasma proteins produced by the immune system as a response to parasitic invasion, can be divided into five classes, IgG, IgA, IgM, IgD, and IgE. Human IgG (HIgG) has an average concentration of 12 mg / ml in adult blood, and encompasses 75% of all human immunoglobulins. HIgG consists of four subclasses IgG1, 2, 3, and 4. Among these subclasses, IgG1 is the most dominant protein, making up anywhere between 43 to 75% of all the HIgG (J. Harris, Blood Separation and Plasma Fractionation, John Wiley & Sons, New York, 325p (1990)). The HIgG backbone consists of two identical “heavy” chains and two identical “light” chains (FIG. 1). Disul...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53A61K39/395C07K7/06C40B40/10
CPCB01D15/3809B01J20/286C07K7/06G01N33/6854C07K1/22
Inventor CARBONELL, RUBENYANG, HAIOUWANG, GUANGQUANGURGEL, PATRICK
Owner NORTH CAROLINA STATE UNIV
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